Stages of Viral Infection at the Cellular Level
Viral Interactions with the Cell Surface and Cell Entry
All viruses must overcome the barrier posed by the cell's plasma membrane in order to deliver their payload of nucleic acid into the cell. Infection is initiated by attachment of the virus to the cell surface. Various cellular proteins, carbohydrates, and lipids (e.g., heparan sulfate proteoglycans, sialic acids, and lectins) can act as attachment factors that concentrate viruses at the cell surface through relatively weak or nonspecific interactions with viral surface proteins. Higher-affinity binding of viral surface proteins to specific cell-surface proteins, or receptors (see Table 120-1), is more critical for viral infection. Receptor binding is often augmented by interaction of viral surface proteins with other cell- surface proteins, or co-receptors, important for various aspects of virus entry. Receptors and co-receptors are important determinants of the cell types and species that a virus can infect. For example, the HIV envelope glycoprotein binds to the T cell surface protein CD4 and then engages one of several chemokine receptors that are co-receptors for the virus. Epstein-Barr virus (EBV) glycoprotein gp350 binds to the B lymphocyte complement receptor CD21 and then uses major histocompatibility complex (MHC) class II molecules as a co-receptor.
Viruses use different strategies to penetrate the cell membrane. Some enveloped viruses use membrane fusion to deliver their contents into the cytoplasm. In general, a trigger (e.g., receptor binding) induces a conformational change that allows the viral surface protein to extend into the cell membrane, bringing the virus and cell membrane into close proximity and thereby enabling fusion and formation of a pore through which the viral nucleocapsid can be delivered into the cytoplasm. Nonenveloped viruses and some enveloped viruses cannot use direct membrane fusion at the plasma membrane and are internalized by endocytosis. The low pH in endosomes can trigger viral membrane or capsid fusion with the endocytic membrane. Conformational changes in nonenveloped capsids can lead to endosomal membrane penetration and release of viral nucleic acid into the cytoplasm.
Influenza virus provides a well-studied example of the effect of low pH on viral penetration. Influenza hemagglutinin mediates adsorption, receptor aggregation, and endocytosis. In low-pH endosomes, changes in conformation of the hemagglutinin expose amphipathic domains that interact chemically with the cell membrane and initiate fusion of the virus and cell membranes. For influenza virus, the M2 membrane protein plays a key role in the uncoating of the viral envelope by providing an ion channel in the envelope.
The fusion of viral proteins with cell membranes is a crucial step in viral infection. The hydrophobic interactions required for fusion can be susceptible to chemical inhibition or blockade. The HIV envelope glycoprotein gp120 is associated with gp41 on the viral surface. Binding of HIV gp120 to CD4 and chemokine receptors results in a conformational change, allowing gp41 to initiate cell membrane fusion. Enfuvirtide is a small-peptide drug derived from gp41 that binds to gp41 and prevents the conformational change required for fusion. Maraviroc prevents virus entry by binding to CCR5, blocking interaction with gp120, and preventing fusion triggering.
Viral Gene Expression and Replication
After uncoating and release of viral nucleoprotein into the cytoplasm, the viral genome is transported to a site for expression and replication. In order to produce infectious progeny, viruses must (1) produce proteins necessary to replicate their nucleic acid, (2) produce structural proteins, and (3) assemble the nucleic acid and proteins into progeny virions. Different viruses use different strategies and gene repertoires to accomplish these goals. DNA viruses, except for poxviruses, replicate their nucleic acid and assemble into nucleocapsid complexes in the cell nucleus. RNA viruses, except for influenza viruses, transcribe and replicate their nucleic acid and assemble entirely in the cytoplasm. Thus, the replication strategies of DNA and RNA viruses are presented separately below. Positive-strand and negative-strand RNA viruses are discussed separately. Medically important viruses of each group are used for illustrative purposes.
Positive-Strand RNA Viruses
Medically important positive-strand RNA viruses include picornaviruses, flaviviruses, togaviruses, caliciviruses, and coronaviruses. Genomic RNA from positive-strand RNA viruses is released into the cytoplasm without associated enzymes. Cell ribosomes recognize and associate with an internal ribosome entry sequence in the viral RNA and translate a polyprotein. Protease components of the polyprotein cleave out the viral RNA polymerase and other viral proteins necessary for replication. Antigenomic RNA is then transcribed from the genomic RNA template. Positive-strand genomes and mRNAs are next transcribed from the antigenomic RNA by the viral RNA polymerase and are translated into capsid proteins. Genomic RNA is encapsidated in the cytoplasm as the infected cell undergoes lysis.
Negative-Strand RNA Viruses
Medically important negative-strand RNA viruses include rhabdoviruses, filoviruses, paramyxoviruses, orthomyxoviruses, and bunyaviruses. The genomes of negative-strand viruses are frequently segmented. Negative-strand RNA virus genomes are released into the cytoplasm with an associated RNA polymerase and one or more polymerase accessory proteins. The viral RNA polymerase transcribes messenger RNAs (mRNAs) as well as full-length antigenomic RNA, which is the template for genomic RNA replication. Viral mRNAs encode the viral RNA polymerase and accessory factors as well as viral structural proteins. Except for influenza virus, which transcribes its mRNAs and antigenomic RNAs in the cell's nucleus, negative-strand RNA viruses replicate entirely in the cytoplasm. All negative-strand RNA viruses, including influenza viruses, assemble in the cytoplasm.
Double-Strand Segmented RNA Viruses
Double-strand RNA viruses are taxonomically grouped in the family Reoviridae. The medically important viruses in this group are rotaviruses and Colorado tick fever virus. Reovirus genomes have 10–12 RNA segments. Reovirus particles contain an RNA polymerase complex. These viruses replicate and assemble in the cytoplasm.
Medically important DNA viruses include parvoviruses, papovaviruses [e.g., human papillomaviruses (HPVs) and polyomaviruses], adenoviruses, herpesviruses, and poxviruses. Most DNA virus genomes enter the cell's nucleus and are transcribed by cellular RNA polymerase II. For example, after receptor binding and fusion with plasma membranes or endocytic vesicle membranes, herpesvirus nucleocapsids are released into the cytoplasm along with tegument proteins. The nucleocapsid is transported along microtubules to a nuclear pore. Capsids then release DNA into the nucleus.
DNA virus transcription and mRNA processing depend on both viral and cellular proteins. For herpes simplex virus (HSV), a viral tegument protein enters the nucleus and activates immediate-early genes, the first genes expressed after infection. Transcription of immediate-early genes requires the viral tegument protein and cell transcription factors. HSV becomes nonreplicating, or latent, in neurons because essential cell transcription factors for viral immediate-early gene expression are docked in the cytoplasm in neurons. Heat shock or other cell stresses can cause these cell factors to enter the nucleus, activate viral gene expression, and initiate replication. This information explains HSV-1 latency in neurons and activation of lytic infection.
For adenoviruses and herpesviruses, immediate-early gene transcription results in expression of early proteins necessary for viral DNA replication. Viral DNA synthesis is required to turn on late gene expression and production of viral structural components. The HPVs, polyomaviruses, and parvoviruses are not dependent on transactivators encoded from the viral genome for early-gene transcription. Instead, their early genes have upstream enhancing elements that bind cell transcription factors. The early genes encode proteins that are necessary for viral DNA synthesis and late-gene transcription. DNA virus late genes encode structural proteins necessary for viral assembly and for viral egress from the infected cell. Late-gene transcription is continuously dependent on DNA replication. Therefore, inhibitors of DNA replication also stop late-gene transcription.
Each DNA virus family uses unique mechanisms for replicating its DNA. Adenovirus and herpesvirus DNAs are linear in the virion. Adenovirus DNA remains linear in infected cells and replicates as a linear genome, using an initiator protein-DNA complex. In contrast, herpesvirus DNA circularizes in the infected cell, and genomes replicate into linear concatemers through a "rolling-circle"mechanism. Full-length DNA genomes are cleaved and packaged into virus. Herpesviruses encode a DNA polymerase and at least six other viral proteins necessary for viral DNA replication. These viruses also encode enzymes that increase the deoxynucleotide triphosphate pools. HPV and polyomavirus DNAs are circular both within the virus and in infected cells. These genomes are reproduced by cellular DNA replication enzymes and remain circular through replication and packaging. HPV and polyomavirus early proteins are necessary for latent and lytic viral DNA replication. Early viral proteins stimulate cells to remain in cycle, facilitating viral DNA replication.
Parvoviruses have negative single-strand DNA genomes and are the smallest DNA viruses. Their genomes are half the size of the papovavirus genomes and include only two genes. The replication of autonomous parvoviruses, such as B19, depends on cellular DNA replication and requires the virus-encoded Rep protein. Other parvoviruses, such as adeno-associated virus (AAV), are not autonomous and require helper viruses of the adenovirus or herpesvirus family for their replication. AAV is being used as a potentially safe human gene therapy vector because its replication protein causes integration at a single chromosome site. The small genome size limits the range of proteins that can be expressed from AAV vectors.
Poxviruses are the largest DNA viruses. They are unique among DNA viruses in replicating and assembling in the cytoplasm. To accomplish cytoplasmic replication, poxviruses encode transcription factors, an RNA polymerase II orthologue, enzymes for RNA capping, enzymes for RNA polyadenylation, and enzymes for viral DNA synthesis. Poxvirus DNA also has a unique structure. The double-strand linear DNA is covalently linked at the ends; the packaged genome is therefore a covalently closed single-strand circle. In addition, there are inverted repeats at the ends of the linear DNA. During DNA replication, the genome is cleaved within the terminal inverted repeat, and the inverted repeats self-prime complementary-strand synthesis by the virus-encoded DNA polymerase. Like herpesviruses, poxviruses encode several enzymes that increase deoxynucleotide triphosphate precursor levels and thus facilitate viral DNA synthesis.
Viruses that Use Both RNA and DNA Genomes in Their Life Cycle
Retroviruses, including HIV, are RNA viruses that use a DNA intermediate to replicate their genomes; hepatitis B virus (HBV) is a DNA virus that uses an RNA intermediate to replicate its genome. Thus these viruses are not purely RNA or DNA viruses. Retroviruses are enveloped RNA viruses with two identical sense-strand genomes and associated reverse transcriptase and integrase enzymes. Retroviruses differ from all other viruses in that they reverse-transcribe themselves into partially duplicated double-strand DNA copies and then routinely integrate into the host genome as part of their replication strategy. The fact that remnants and even complete copies of simple retroviral DNA are integrated into the human genome raises the possibility of replication-competent simple human retroviruses. However, replication has not been documented or associated with any disease. Integrated, replication-competent retroviral DNAs are also present in many animal species, such as pigs. These porcine retroviruses are a potential cause for concern in xenotransplantation because retroviral replication could cause disease in humans.
Cellular RNA polymerase II and transcription factors regulate transcription from the integrated provirus DNA genome. Some retroviruses also encode for regulators of transcription and RNA processing, such as Tax and Rex in human T lymphotropic virus (HTLV) types I and II. HIV-1 and HIV-2 have orthologous Tat and Rev genes as well as the additional accessory proteins Vpr, Vpu, and Vif, which are important for efficient infection and immune escape. Full-length proviral transcripts are made from a promoter in the viral terminal repeat and serve as both genomic RNAs that will be packaged in the nucleocapsids and differentially spliced mRNAs that encode for the viral Gag protein, polymerase/integrase protein, and envelope glycoprotein. The Gag protein includes a protease that cleaves it into several components, including a viral matrix protein that coats the viral RNA. Viral RNA polymerase/integrase, matrix protein, and cellular tRNA are key components of the viral nucleocapsid. The HIV reverse transcriptase, integrase, and Gag protease are important targets for inhibition of HIV replication.
HBV replication is unique in several respects. HBV has a partially double-strand DNA genome that is repaired to a fully double-strand circular DNA by the virion polymerase upon entry into an infected cell. Viral mRNAs are transcribed from the closed circular viral episome by cellular RNA polymerase II and are translated to produce viral proteins including core protein, surface antigen, and polymerase. In addition, a full-genome-length mRNA is packaged into viral core particles in the cytoplasm of infected cells as an intermediate for viral DNA replication. This RNA associates with the viral polymerase, which also has reverse transcriptase activity, to convert the full-length encapsidated RNA genome into partially double-strand DNA. HBV is believed to mature by budding through the cell's plasma membrane, which has been modified by the insertion of viral surface antigen protein.
Viral Assembly and Egress
For most viruses, nucleic acid and structural protein synthesis is accompanied by the assembly of protein and nucleic acid complexes. The assembly and egress of mature infectious virus mark the end of the eclipse phase of infection, during which infectious virus cannot be recovered from the infected cell. Nucleic acids from RNA viruses and poxviruses assemble into nucleocapsids in the cytoplasm. For all DNA viruses except poxviruses, viral DNA assembles into nucleocapsids in the nucleus. In general, the capsid proteins of viruses with icosahedral nucleocapsids can self-assemble into densely packed and highly ordered capsid structures. Herpesviruses require an assemblin protein as a scaffold for capsid assembly. Viral nucleic acid then spools into the assembled capsid. For herpesviruses, a full unit of the viral DNA genome is packaged into the capsid, and a capsid-associated nuclease cleaves the viral DNA at both ends. In the case of viruses with helical nucleocapsids, the protein component appears to assemble around the nucleic acid, which contributes to capsid organization.
Viruses must egress from the infected cell and not bind back to their receptor(s) on the outer surface of the plasma membrane. Viruses can acquire envelopes from cytoplasmic membranes or by budding through the cell's plasma membrane. Excess viral membrane glycoproteins are synthesized to saturate cell receptors and facilitate separation of the virus from the infected cell. Some viruses encode membrane proteins with enzymatic activity for receptor destruction. Influenza virus, for example, encodes a glycoprotein with neuraminidase activity. Neuraminidase destroys sialic acid on the infected cell's plasma membrane so that newly released virus does not get stuck to the dying cell. Herpesvirus nucleocapsids acquire an initial envelope by assembling in the nucleus and then budding through the nuclear membrane into the endoplasmic reticular space. The initially enveloped herpesvirus is then de-enveloped and released from the cell either by exocytosis or by re-envelopment at the plasma membrane. Nonenveloped viruses depend on the death and dissolution of the infected cell for their release.
Fidelity of Viral Replication
Hundreds or thousands of progeny may be produced from a single virus-infected cell. Many particles partially assemble and never mature into virions. Many mature-appearing virions are imperfect and have only incomplete or nonfunctional genomes. Despite the inefficiency of assembly, a typical virus-infected cell releases 10–1000 infectious progeny. Some of these progeny may contain genomes that differ from those of the virus that infected the cell. Smaller, "defective" viral genomes have been noted with the replication of many RNA and DNA viruses. Virions with defective genomes can be produced in large numbers through packaging of incompletely synthesized nucleic acid. Adenovirus packaging is notoriously inefficient, and a high ratio of particle to infectious virus may limit the amount of recombinant adenovirus that can be administered for gene therapy since the immunogenicity of defective particles may contribute to adverse effects.
Changes in viral genomes can lead to mutant viruses of medical significance. In general, viral nucleic acid replication is more error-prone than cellular nucleic acid replication. RNA polymerases and reverse transcriptases are significantly more error-prone than DNA polymerases. Mutations can also be introduced into the HIVgenome by APOBEC3G, a cellular protein that is packaged in the virion. APOBEC3G deaminates cytidine in the virion RNA to uridine. When reverse transcriptase subsequently uses the altered virion RNA as a template in the infected cell, a guanosine-to-adenosine mutation is introduced into the proviral DNA. Mutations resulting in less efficient viral growth, or fitness, may be detrimental to the virus. HIV-encoded Vif blocks APOBEC3G activity in the virion, inhibiting the debilitating effects of hypermutation on genetic integrity. Nevertheless, mutations resulting in evasion of the host immune response or resistance to antiviral drugs are preferentially selected in patients, with the consequent perpetuation of infection. Viral genomes can also be altered by recombination or reassortment between two related viruses in a single infected cell. While this occurrence is unusual under most circumstances of natural infection, the genome changes can be substantial and can significantly alter virulence or epidemiology. Reassortment of the avian or mammalian influenza A hemagglutiningene into a human influenza background can result in the emergence of new epidemic or pandemic influenza A strains.
Viral Genes Not Required for Viral Replication
Viruses frequently have genes encoding proteins that are not directly involved in replication or packaging of the viral nucleic acid, in virion assembly, or in regulation of the transcription of viral genes involved in those processes. Most of these proteins fall into five classes: (1) proteins that directly or indirectly alter cell growth; (2) proteins that inhibit cellular RNA or protein synthesis so that viral mRNA can be efficiently transcribed or translated; (3) proteins that promote cell survival or inhibit apoptosis so that progeny virus can mature and escape from the infected cell; (4) proteins that inhibit the host interferon response; and (5) proteins that downregulate host inflammatory or immune responses so that viral infection can proceed in an infected person to the extent consistent with the survival of the virus and its efficient transmission to a new host. More complex viruses of the poxvirus or herpesvirus family encode many proteins that serve these functions. Some of these viral proteins have motifs similar to those of cellular proteins, while others are quite novel. Virology has increasingly focused on these more sophisticated strategies evolved by viruses to permit the establishment of long-term infection in humans and other animals. These strategies often provide unique insights into the control of cell growth, cell survival, macromolecular synthesis, proteolytic processing, immune or inflammatory suppression, immune resistance, cytokine mimicry, or cytokine blockade.
MicroRNAs (miRNAs) are small noncoding RNAs that can regulate gene expression at the posttranscriptional level by targeting–and usually silencing–mRNAs. MiRNAs were initially discovered in plants and plant viruses, where they alter expression of cell defensins. Herpesviruses are especially rich in miRNAs; for example, at least 23 miRNAs have been identified in EBV and 11 in cytomegalovirus (CMV). Adenovirus and polyomavirus miRNAs have also been described. Increasing data indicate that animal viruses encode miRNAs to alter the growth and survival of host cells and the innate and acquired immune responses.
The concept of host range was originally based on the cell types in which a virus replicates in tissue culture. For the most part, the host range is limited by specific cell-surface proteins required for viral adsorption or penetration—i.e., to the cell types that express receptors or co-receptors for a specific virus. Another common basis for host-range limitation is the degree of transcriptional activity from viral promoters in different cell types. Most DNA viruses depend not only on cellular RNA polymerase II and the basal components of the cellular transcription complex but also on activated components and transcriptional accessory factors, both of which differ among differentiated tissues, among cells at various phases of the cell cycle, and between resting and cycling cells. APOBEC3G, an important cell restriction factor for HIV infection, hypermutates viral RNA. The balance between HIV Vif and APOBEC3G is an important determinant of HIV-1 infection.
The importance of host range factors is illustrated by the effects of specific host determinants that limit the replication of influenza virus with avian or porcine hemagglutinins in humans. These viral proteins have adapted to bind avian or porcine sialic acids, and spread of avian or porcine influenza viruses in human populations is limited by their ability to infect human cells.
Viral Cytopathic Effects and Inhibitors of Apoptosis
The replication of almost all viruses has adverse effects on the infected cell, inhibiting cellular synthesis of DNA, RNA, or proteins through efficient competition for key substrates and enzymatic processes. These general inhibitory effects enable viruses to nonspecifically limit components of innate host resistance, such as interferon (IFN) production. Viruses can specifically inhibit host protein synthesis by attacking a component of the translational initiation complex–frequently, a component that is not required for efficient translation of viral RNAs. Poliovirus protease 2A, for example, cleaves a cellular component of the complex that ordinarily facilitates translation of cellular mRNAs by interacting with their cap structure. Poliovirus RNA is efficiently translated without a cap since it has an internal ribosome entry sequence. Influenza virus inhibits the processing of mRNA by snatching cap structures from nascent cellular RNAs and using them as primers in the synthesis of viral mRNA. HSV has a virion tegument protein that inhibits cellular mRNA translation.
Apoptosis is the expected consequence of virus-induced inhibition of cellular macromolecular synthesis and viral nucleic acid replication. While the induction of apoptosis may be important for the release of some viruses (particularly nonenveloped viruses), many viruses have acquired genes or parts of genes that enable them to forestall infected-cell death. This delay increases the yield from viral replication. Adenoviruses and herpesviruses encode analogues of the cellular Bc12 protein, which blocks mitochondrial enhancement of proapoptotic stimuli. Poxviruses and some herpesviruses also encode caspase inhibitors. Many viruses, including HPVs and adenoviruses, encode proteins that inhibit p53 or its downstream proapoptotic effects.